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UNKNOWN LAB REPORT
The human body contains as much as 10 times as many bacteria as human cells. These bacteria inhabit our gastrointestinal tract, mouth, skin, and most other areas of the body. Our normal flora are harmless to us within normal quantitative ranges, and are even necessary for digestion and normal bodily functions, but it is important to be aware of both our normal flora and pathogenic bacteria alike. This study highlights some of the capabilities and qualities of bacteria humans contact often and how they compare and contrast with each other.
MATERIALS AND METHODS
The two different bacteria were first given simultaneously in a nutrient agar (NA) broth, labeled as “Unknown Bacteria #104.” The professor gave the initial information that there was one Gram-positive and one Gram-negative bacteria each in the NA broth. Using procedures in the supplied lab manual, the bacteria were isolated to their own nutrient agar plates, and then identified using a variety of methods, including a gram stain and several selective and differential tests. These methods will be expanded upon in detail in this report.
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The following tests were conducted during the identification of both unknown bacteria:
- Mannitol Salt Agar (MSA)
- Simmon’s Citrate
- Methyl Red
- Eosin-Methylene Blue (EMB)
To first isolate the bacteria, a streak test in a NA plate was performed with the intent of isolating a pure culture. After 48 hours of incubation, only one bacteria grew in the original NA plate. From a pure culture in the original plate, an additional streak test in a NA plate was conducted to ensure no contamination from the original bacteria that did not grow. After the second NA plate with the pure culture had been incubated for seven (7) days (168 hours), bacteria from this plate was used to conduct a gram stain to discover if the isolated bacteria was Gram-negative or Gram-positive. The initial gram stain, which was conducted as instructed in the lab manual, showed the isolated bacteria was a Gram-negative rod.
In order to isolate the Gram-positive bacteria, a streak test from the original unknown NA tube was conducted on Mannitol Salt Agar (MSA), which inhibits the growth of Gram-negative bacteria. After five (5) days of incubation, the MSA showed no growth, so a second MSA test was done to ensure the negative result. The second MSA test was also negative for all growth. At this time, the priority was decided to be identifying the Gram-negative bacteria.
Upon isolating the unknown Gram-negative rod, a series of differential mediums were used to hypothesize the unknown Gram-negative and Gram-positive bacteria.
Following determining the isolated, unknown bacteria was Gram-negative, a urea test was conducted to further exclude potential bacteria possibilities. The initial urea test of the Gram-negative bacteria was negative, suggesting the unknown Gram-negative bacteria does not contain the enzyme urease, and is therefore Escherichia coli, Enterobacter aerogenes, or Pseudomonas aeruginosa. At this time a second gram stain was conducted to ensure this isolated bacteria was certainly Gram-negative. The second gram stain also showed the bacteria to be a Gram-negative rod, proving the initial test accurate. After confirming the isolated bacteria was Gram-negative, a Simmon’s Citrate test was conducted on the Gram-negative bacteria to further differentiate the bacteria. The Simmon’s Citrate test was positive, indicating that the unknown Gram-negative bacteria produced citrate permease, which allows it to take in citrate and convert it to pyruvate. This positive result narrowed down the possibilities to E. aerogenes and P. aeruginosa. To narrow down the bacteria to a single possibility, several tests were conducted. The first were Methyl Red and Voges-Proskauer (MR/VP) tests. The same medium is used for both tests and, after incubation; separate reactants are added to the inoculated broths to test for acid and acetoin production, respectively.
The results for the unknown bacteria were negative for both MR and VP tests, suggesting the unknown Gram-negative bacteria be P. aeruginosa. To confirm this suspicion, gelatin and oxidase tests were conducted. This test was chosen to differentiate between P. aeruginosa and E. aerogenes because P. aeruginosa, the suspected bacteria, should have a positive result in both tests, and E. aerogenes, a negative result in both tests. While the oxidase test showed an only slightly positive result, it was still accepted as a positive result. The gelatin test was also positive, proving that the unknown bacteria is able to hydrolyze gelatin via the extracellular enzyme gelatinase. These positive test results suggest the unknown Gram-negative bacteria is P. aeruginosa.
While the results of the tests show the Gram-negative bacteria to be P. aeruginosa, the professor advises that this conclusion is incorrect. The unknown Gram-negative bacteria were E. aerogenes. To confirm this result, an eosin-methylene blue (EMB) test was conducted. After seven (7) days of incubation, the EMB agar had pink colonies, verifying the unknown bacteria ferments lactose and has weak acid end products, and confirms the unknown Gram-negative bacteria was indeed E. aerogenes. The inconsistencies between the presumed bacteria and actual bacteria were likely caused by three (3) tests. First, the VP test likely had a false negative result. Second, the oxidase test which showed a slight positive result was likely actually a negative result. Finally, the gelatin test was either contaminated or the test was performed incorrectly, producing a false positive result.
During the course of identifying the unknown Gram-negative bacteria, a total of three (3) MBA tests were conducted to isolate the unknown Gram-positive bacteria- all of which resulted in negative growth.
After the identification of the unknown Gram-negative bacteria, precedence was given to identifying the unknown Gram-positive bacteria. Since the unknown Gram-positive bacteria from the initial NA broth were unable to be isolated, a pure culture of different Gram-positive bacteria was obtained from the professor. Upon receiving this new pure culture, several tests were conducted simultaneously to accelerate the identification of these bacteria. One of these tests was the Simmon’s Citrate test, which would tell if the unknown Gram-positive bacteria were a rod (positive result) or a cocci (negative result). The result of this test was negative, suggesting the bacteria be Staphylococcus aureus, S. epidermidis or Enterococcus faecalis.
To narrow down the possibilities, a catalase test was conducted to select for E. faecalis. The results to this test were positive, which suggests the unknown Gram-positive bacteria is capable of producing an enzyme which converts hydrogen peroxide to water and molecular oxygen, and is either S. aureus or S. epidermidis. To differentiate between these two bacteria, a urea test was done. The negative result of this test means the unknown Gram-positive bacteria does not produce urease to break down urea, and is S. aureus. Preferably a final test would be conducted to select for S. aureus with a positive result, but there were no tests readily available which would select for this bacteria over S. epidermidis with a positive result for S. aureus.
The results obtained in these tests suggest the unknown Gram-positive to be S. aureus, but the professor indicated the unknown bacteria to be Bacillus cereus. Unfortunately time did not allow for additional tests to conclude where the mistake or false result was made, but I believe the error occurred during the Simmon’s Citrate test. If time would have allowed, an additional Simmon’s Citrate test would have been conducted, as well as an oxidase and casein test, both of which are selective for B. cereus.
Bacillus cereus is an aerobic spore-forming bacterium that is commonly found in soil, on vegetables, and in many raw and processed foods. B. cereus food poisoning may occur when food is prepared and held without adequate refrigeration for several hours before serving. Foods incriminated in past outbreaks include cooked meat and vegetables, boiled or fried rice, vanilla sauce, custards, soups, and raw vegetable sprouts. (3) B. cereus is a type of bacteria that produces toxins. These toxins can cause two types of illness: one type characterized by diarrhea and the other, called emetic toxin, by nausea and vomiting (1). Although no specific complications have been associated with the diarrheal and vomiting toxins produced by B. cereus, other clinical manifestations of B. cereus invasion or contamination have been observed. They include bovine mastitis, severe systemic and pyogenic infections, gangrene, septic meningitis, cellulitis, panophthalmitis, lung abscesses, infant death, and endocarditis. (2)